The present invention relates to the determination of functional HCV virus genomic RNA sequences, to construction of infectious HCV DNA clones, and to use of the clones, or their derivatives, in therapeutic, vaccine, and diagnostic applications. The invention is also directed to HCV vectors, e.g., for gene therapy or gene vaccines.
After the development of diagnostic tests for hepatitis A virus and hepatitis B virus, an additional agent, which could be experimentally transmitted to chimpanzees [Alter et al., Lancer 1, 459-463 (1978); Hollinger et al., Intervirology 10, 60-68 (1978): Tabor et al., Lancet 1, 463-466 (1978)], became recognized as the major cause of transfusion-acquired hepatitis. cDNA clones corresponding to the causative non-A non-B (NANB) hepatitis agent, called hepatitis C virus (HCV), were reported in 1989 [Choo et al., Science 244, 359-362 (1989)]. This breakthrough has led to rapid advances in diagnostics, and in our understanding of the epidemiology, pathogenesis and molecular virology of HCV (see Houghton et al., Curr Stud Hematol Blood Transfus 61, 1-11 (1994) for review). Evidence of HCV infection is found throughout the world, and the prevalence of HCV-specific antibodies ranges from 0.4-2% in most countries to more than 14% in Egypt [Hibbs et al., J. Inf. Dis. 168, 789-790 (1993)]. Besides transmission via blood or blood products, or less frequently by sexual and congenital routes, sporadic cases, not associated with known risk factors, occur and account for more than 40% of HCV cases [Alter et al., J. Am. Med. Assoc. 264, 2231-2235 (1990); Mast and Alter, Semin. Virol. 4, 273-283 (1993)]. Infections are usually chronic [Alter et al., N. Eng. J. Med. 327, 1899-1905 (1992)], and clinical outcomes range from an in apparent carrier state to acute hepatitis, chronic active hepatitis, and cirrhosis which is strongly associated with the development of hepatocellular carcinoma.
Although interferon (IFN)-xcex1 has been shown to be useful for tile treatment of a minority of patients with chronic HCV infections (Davis et al., N. Engl. J. Med 321, 1501-1506 (1989); DiBisceglie et al., New Engl. J. Med 321, 1506-1510 (1989)) and subunit vaccines show some promise in the chimpanzee model [Choo et al., Proc. Natl. Acad. Sci. USA 91, 1294-1298 (1994)], future efforts are needed to develop more effective therapies and vaccines. The considerable diversity observed among different HCV isolates [for review, see Bukh et al., Sem. Liver Dis. 15, 41-63 (1995)], the emergence of genetic variants in chronically infected individuals [Enomoto et al., J. Hepatol. 17, 415-416 (1993); Hijikata et al., Biochem. Biophys. Res. Comm. 175, 220-228 (1991); Kato et al., Biochem. Biophys. Res. Comm. 189, 119-127 (1992); Kato et al., J. Virol. 67, 3923-3930 (1993); Kurosaki et al., Hepatology 18, 1293-1299 (1993); Lesniewski et al., J. Med. Virol. 40, 150-156 (1993): Ogata et al., Proc. Natl. Acad. Sci. USA 88, 3392-3396 (1991); Weiner et al., Virology 180, 842-848 (1991); Weiner et al., Proc. Natl Acad. Sci. USA 89, 3468-3472 (1992)], and the lack of protective immunity elicited after HCV infection [Farci et al., Science 258, 135-140 (1992); Prince et al., J. Infect. Dis. 165, 438-443 (1992)] present major challenges towards these goals.
Classification. Based on its genome structure and virion properties, HCV has been classified as a separate genus in the flavivirus family, which includes two other genera: the flaviviruses (e.g., yellow fever (YF) virus) and the animal pestiviruses (e.g., bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV)) [Francki et al., Arch. Virol. Suppl. 2, 223 (1991)]. All members of this family have enveloped virions that contain a positive-strand RNA genome encoding all known virus-specific proteins via translation of a single long open reading frame (ORF).
Structure and physical properties of the virion. Little information is available on the structure and replication of HCV. Studies have been hampered by the lack of a cell culture system able to support efficient virus replication and the typically low titers of infectious virus present in serum. The size of infectious virus, based on filtration experiments, is between 30-80 nm [Bradley et al., Gastroenterology 88, 773-779 (1985); He et al., J. Infect. Dis. 156, 636-640 (1987); Yuasa et al., J. Gen. Virol. 72, 2021-2024 (1991)]. Initial measurements of the buoyant density of infectious material in sucrose yielded a range of values, with the majority present in a low density pool of  less than 1.1 g/ml [Bradley et al., J. Med. Virol. 34, 206-208 (1991)]. Subsequent studies have used RT/PCR to detect HCV-specific RNA as an indirect measure of potentially infectious virus present in sera from chronically infected humans or experimentally infected chimpanzees. From these studies, it has become increasingly clear that considerable heterogeneity exists between different clinical samples, and that many factors can affect the behavior of particles containing HCV RNA [Hijikata et al., J. Virol. 67, 1953-1 958 (1993); Thomssen et al., Med. Microbiol. Immunol. 181, 293-300 (1992)]. Such factors include association with immunoglobulins [Hijikata et al., (1993) supra] or low density lipoprotein [Thomssen et al., 1992, supra; Thomssen et al., Med. Microbiol. Immunol. 182, 329-334 (1993)]. In highly infectious acute phase chimpanzee serum, HCV-specific RNA is usually detected in fractions of low buoyant density (1.03-1.1 g/ml) [Carrick et al., J. Virol. Meth. 39, 279-289 (1992); Hijikata et al., (1993) supra]. In other samples, the presence of HCV antibodies and formation of immune complexes correlate with particles of higher density and lower infectivity [Hijikata et al., (1993) supra]. Treatment of particles with chloroform, which destroys infectivity [Bradley et al., J. Infect. Dis. 148, 254-265 (1983); Feinstone et al., Infect Immun. 41, 816-821 (1983)], or with nonionic detergents, produced RNA containing particles of higher density (1.17-1.25 g/ml) believed to represent HCV nucleocapsids [Hijikata et al., (1993) supra; Kanto et al., Hepatology 19, 296-302 (1994); Miyamoto et al., J. Gen. Virol. 73, 715-718 (1992)].
There have been reports of negative-sense HCV-specific RNAs in sera and plasma [see Fong et al., Journal of Clinical Investigation 88:1058-60 (1991)]. However, it seems unlikely that such RNAs are essential components of infectious particles since some sera with high infectivity can have low or undetectable levels of negative-strand RNA [Shimizu et al., Proc. Natl. Acad. Sci. USA 90: 6037-6041 (1993)].
The virion protein composition has not been rigorously determined, but putative HCV structural proteins include a basic C protein and two membrane glycoproteins,E1 and E2.
HCV replication. Early events in HCV replication are poorly understood. Cellular receptors for the HCV glycoproteins have not been identified. The association of some HCV particles with beta-lipoprotein and immunoglobulins raises the possibility that these host molecules may modulate virus uptake and tissue tropism. Studies examining HCV replication have been largely restricted to human patients or experimentally inoculated chimpanzees. In the chimpanzee model, HCV RNA is detected in the serum as early as three days post-inoculation and persists through the peak of serum alanine aminotransferase (ALT) levels (an indicator of liver damage) [Shimizu et al., Proc. Natl. Acad. Sci. USA 87: 6441-6444 (1990)]. The onset of viremia is followed by the appearance of indirect hallmarks of HCV infection of the liver. These include the appearance of a cytoplasmic antigen [Shimizu et al., (1990) supra] and ultrastructural changes in hepatocytes such as the formation of microtubular aggregates for which HCV previously was referred to as the chloroform-sensitive xe2x80x9ctubule forming agentxe2x80x9d or xe2x80x9cTFAxe2x80x9d [reviewed by Bradley, Prog. Med. Virol. 37: 101-135 (1990)]. As shown by the appearance of viral antigens [Blight et al., Amer. J. Path. 143: 1568-1573 (1993); Hiramatsu et al., Hepatology 16: 306-311 (1992); Krawczynski et al., Gastroenterology 103: 622-629 (1992); Yamada et al., Digest. Dis. Sci. 38: 882-887 (1993)] and the detection of positive and negative sense RNAs [Fong et al., (1991)supra; Gunji et al., Arch. Virol. 134: 293-302(1994): Haruna et al., J. Hepatol. 18: 96-100 (1993); Lamas et al., J. Hepatol. 16: 219-223 (1992); Nouri Aria et al., J. Clin. Inves. 91: 2226-34 (1993); Sherker et al., J. Med. Virol. 39: 91-96 (1993); Takehara et al., Hepatology 15: 387-390 (1992); Tanaka et al., Liver 13: 203-208 (1993)], hepatocytes appear to be a major site of HCV replication, particularly during acute infection [Negro et al., Proc. Natl. Acad. Sci. USA 89: 2247-2251 (1992)]. In later stages of HCV infection the appearance of HCV-specific antibodies, the persistence or resolution of viremia, and the severity of liver disease, vary greatly both in the chimpanzee model and in human patients. Although some liver damage may occur as a direct consequence of HCV infection and cytopathogenicity, the emerging consensus is that host immune responses, in particular virus-specific cytotoxic T lymphocytes, may play a more dominant role in mediating cellular damage.
It has been speculated that HCV may also replicate in extra-hepatic reservoir(s). In some cases, RT/PCR or in situ hybridization has shown an association of HCV RNA with peripheral blood mononuclear cells including T-cells, B-cells, and monocytes reviewed in Blight and Gowans, Viral Hepatitis Rev. 1: 143-155 (1995)]. Such tissue tropism could be relevant to the establishment of chronic infections and might also play a role in the association between HCV infection and certain immunological abnormalities such as mixed cryoglobulinemia [reviewed by Ferri et al., Eur. J. Clin. Invest. 23: 399-405 (1993)], glomerulonephritis, and rare non-Hodgkin""s B-lymphomnas [Ferri et al., (1993) supra; Kagawa et al., Lancet 341: 316-317 (1993)]. However, the detection of circulating negative strand RNA in serum, the difficulty in obtaining truly strand-specific RT/PCR [Gunji et al., (1994) supra], and the low numbers of apparently infected cells have made it difficult to obtain unambiguous evidence for replication in these tissues in vivo.
Genome structure. Full-length or nearly full-length genome sequences of numerous HCV isolates have been reported [see Lin et al., J. Virol. 68: 5063-5073 (1994a): Okamoto et al., J. Gen. Virol. 75: 629-635 (1994); Sakamoto et al., J. Gen. Virol. 75: 1761-1768 (1994) and citations therein]. Given the considerable genetic divergence among isolates, it is clear that several major HCV genotypes are distributed throughout the world. Those of greatest importance in the U.S. are genotype 1, subtypes 1a and 1b (see below and Ref. Bukh et al., (1995) supra for a discussion of genotype prevalence and distribution). HCV genome RNAs are xcx9c9.6 kilobases in length (FIG. 1). The 5xe2x80x2 NTR is 341-344 bases long and highly conserved. The length of the long ORF varies slightly among isolates, encoding polyproteins of 3010. 3011 or 3033 amino acids. The reported 3xe2x80x2 NTR structures show considerable diversity both in composition and length (28-42 bases), and appear to terminate with poly (U) [see Chen et al., Virology 188:102-113 (1992); Okamoto et al., J. Gen. Virol. 72:2697-2704 (1991); Tokita et al., J. Gen. Virol. 66:1476-83 (1994)] except in one case (HCV-1. type 1a) which appears to contain a 3xe2x80x2 terminal poly (A) tract (Han et al., Proc. Natl. Acad. Sci. USA 88:1711-1715 (1991)). In contrast, our recent analysis suggests that the genome RNA of the H-strain (also type 1a) contains an internal polypyrimidine tract followed by a novel RNA element [patent application Ser. No. 08/520, 678, filed Aug. 29, 1995, and International Patent Application No. PCT/US96/14033, filed Aug. 28, 1996]. The results presented in application Ser. No. 08/520, 678 show that the genome RNA of this type 1a isolate does not terminate with a homopolymer tract as previously thought, but rather with a novel sequence of xcx9c98 bases. Furthermore, this 3xe2x80x2 NTR structure and the novel 3xe2x80x2 terminal element are features common to all HCV genotypes which have thus far been examined [Kolykhalov et al., J. Virol. 70: 3363-3371 (1996); Tanaka et al., Biochem. Biophys. Res. Comm. 215: 744-749 (1996); Tanaka et al., J. Virol. 70:3307-12 (1996); Yamada et al., Virology 223:255-261 (1996)).
Translation and proteolytic processing. Several studies have used cell-free translation and transient expression in cell culture to examine the role of the 5xe2x80x2 NTR in translation initiation [Fukushi er al., Biochem. Biophys. Res. Comm. 199: 425-432 (1994); Tsukiyama-Kohara et al., J. Virol. 66: 1476-1483 (1992): Wang et al., J. Virol. 67: 3338-3344 (1993); Yoo et al., Virology 191: 889-899 (1992)]. This highly conserved sequence contains multiple short AUG-initiated ORFs and shows significant homology with the 5xe2x80x2 NTR region of pestiviruses (Bukh et al., Proc. Natl. Acad. Sci. USA 89: 4942-4946 (1992), Han et al., (1991) supra]. A series of stem-loop structures have been proposed on the basis of computer modeling and sensitivity to digestion by different ribonucleases [Brown et al., Nucl. Acids Res. 20: 5041-5045 (1992); Tsukiyama-Kohara et al., (1992) supra]. The results from several groups indicate that this element functions as an internal ribosome entry site (IRES) allowing efficient translation initiation at the first AUG of the long ORF [Fukushi er al., (1994) supra; Tsukiyama-Kohara et al., (1992) supra; Wang et al., (1993) supra: Yoo et al., (1992) supra]. Some of the predicted features of the HCV and pestivirus IRES elements are similar to one another [Brown et al., (1992) supra]. The ability of this element to function as an IRES suggests that HCV genome RNAs may lack a 5xe2x80x2 cap structure.
The organization and processing of the HCV polyprotein (FIG. 1) appears to be most similar to that of the pestiviruses. At least 10 polypeptides have been identified and the order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. As shown in FIG. 1, proteolytic processing is mediated by host signal peptidase and two HCV-encoded proteinases, the NS2-3 autoproteinase and the NS3-4A serine proteinase [see Rice, In xe2x80x9cFields Virologyxe2x80x9d (B. N. Fields, D. M. Knipe and P. M. Howley, Eds.), Vol. pp. 931-960. Raven Press, New York (1996); Shimotohno et al., J. Hepatol. 22: 87-92 (1995) for reviews]. C is a basic protein believed to be the viral core or capsid protein; E1 and E2 are putative virion envelope glycoproteins; p7 is a hydrophobic protein of unknown function that is inefficiently cleaved from the E2 glycoprotein [Lin et al., (1994a) supra; Mizushima et al., J. Virol. 68: 6215-6222 (1994); Selby et al., Virology 204: 114-122 (1994)], and NS2-NS5B are likely nonstructural (NS) proteins which function in viral RNA replication complexes. In particular besides its N-terminal serine proteinase domain, NS3 contains motifs characteristic of RNA helicases and has been shown to possess an RNA-stimulated NTPase activity [Suzich et al., J. Virol. 67, 6152-6158 (1993)]; NS5B contains the GDD motif characteristic of the RNA-dependent RNA polymerases of positive-strand RNA viruses.
HVC RNA replication. By analogy with flaviviruses, replication of the positive-sense HCV virion RNA is thought to occur via a minus-strand intermediate. This strategy can be described briefly as follows: (i) uncoating of the incoming virus particle releases the genomic plus-strand, which is translated to produce a single long polyprotein that is probably processed co- and post-translationally to produce individual structural and nonstructural proteins: (ii) the nonstructural proteins presumably form a replication complex that utilizes the virion RNA as template for the synthesis of minus strands; (iii) these minus strands in turn serve as templates for synthesis of plus strands, which can be used for additional translation of viral protein, minus strand synthesis, or packaging into progeny virions. Very few details about HCV replication process are available, due to the lack of a good experimental system for virus propagation. Detailed analyses of authentic HCV replication and other steps in the viral life cycle would be greatly facilitated by the development of an efficient system for HCV replication in cell culture.
Many attempts have been made to infect cultured cells with serum collected from HCV-infected individuals, and low levels of replication have been reported in a number of cells types infected by this method, including B-cell [Bertolini et al., Res. Virol. 144: 281-285 (1993); Nakajima et al., J. Virol. 70: 9925-9 (1996); Valli et al., Res. Virol. 146:285-288 (1995)]. T-cell (Kato et al., Biochem. Biophys. Res. Commun. 206:863-9 (1996); Mizutani et al., Biochem. Biophys. Res. Comm. 227:822-826; Mizutani et al., J. Virol. 70: 7219-7223 (1996); Nakajima et al., (1996) supra; Shimizu and Yoshikura, J. Virol. 68: 8406-8408 (1994); Shimizu et al., Proc. Natl. Acad. Sci. USA, 89: 5477-5481 (1992); Shimizu et al., Proc. Natl. Acad. Sci. USA, 90: 6037-6041 (1993)), and hepatocyte [Kato et al., Jpn. J. Cancer Res., 87: 787-92 (1996); Tagawa, J. Gastoenterol and Hepatol., 10: 523-527 (1995)] cell lines, as well as peripheral blood monocular cells (PBMCs) [Cribier et al., J. Gen. Virol., 76: 2485-2491 (1995)], and primary cultures of human fetal hepatocytes [Carloni et al., Arch. Virol. Suppl. 8: 31-39(1993); Cribier et al., (1995) supra; Iacovacci et al., Res. Virol., 144: 275-279 (1993)] or hepatocytes from adult chimpanzees [Lanford et al., Virology 202: 606-14 (1994)]. HCV replication has also been detected in primary hepatocytes derived from a human HCV patient that were infected with the virus in vivo prior to cultivation [Ito et al., J. Gen. Virol. 77: 1043-1054 (1996)] and in the human hepatoma cell line Huh7 following transfection with RNA transcribed in vitro from an HCV-1 cDNA clone [Yoo et al., J. Virol., 69: 32-38 (1995)). The reported observation of replication in cells transfected with RNA derived from the HCV-1 clone was puzzling, since this clone lacks the 3xe2x80x2NTR sequence downstream of the homopolymer tract (see below). The most well-characterized cell-culture systems for HCV replication utilize a B-cell line (Daudi) or T-cell lines persistently infected with retroviruses (HPB-Ma or MT-2) [Kato et al., (1995) supra; Mizutani et al., Biochem Biophys. Res. Comm., 227: 822-826 (1996a); Mizutani et al., (1996) supra; Nakajima et al., (1996) supra; Shimizu and Yoshikura, (1994) supra]; Shimizu, Proc. Natl. Acad. Sci. USA, 90: 6037-6041 (1993)]. HPBMa is infected with an amphotropic murine leukemia virus pseudotype of murine sarcoma virus, while MT-2 is infected with human T-cell lymphotropic virus type I (HTLV-I). Clones (HPBMa10-2 and MT-2C) that support HCV replication more efficiently than the uncloned population have been isolated for the two T-cell lines HPBMa and MT-2 [Mizutani et al. J. Virol. (1996) supra; Shimizu et al., (1993) supra]. However, the maximum levels of RNA replication obtained in these lines or in the Daudi lines after degradation of the input RNA is still only about 5xc3x97104 RNA molecules per 106 cells [Mizutani et al., (1996) supra; Mizutani et al., (1996) supra] or 104 RNA molecules per ml of culture medium (Nakajima et al., (1996) supra]. Although the level of replication is low, long-term infections of up to 198 days in one system [Mizutani et al., Biochem. Biophys. Res. Comm. 227: 822-826 (1996a)] and more than a year in another system [Nakajima et al., (1996) supra] have been documented, and infectious virus production has been demonstrated by serial cell-free or cell-mediated passage of the virus to naive cells.
However, efficient HCV replication has not been observed in any of the cell-culture systems described to date, and all of the groups that have attempted to establish such systems have encountered a number of problems, including the difficulty in distinguishing input RNA from plus strands produced by replication, the false detection of minus strands, and generally low titers of replicated RNA. Thus, despite these advances, more efficient cell-culture systems for HCV propagation are needed for the production of concentrated virus stocks, structural analysis of virion components, and improved analyses of intracellular viral processes, including RNA replication.
Virion assembly and release. This process has not been examined directly, but the lack of complex glycans, the ER localization of expressed HCV glycoproteins [Dubuisson et al., J. Virol. 68: 6147-6160 (1994); Ralston et al., J. Virol. 67: 6753-6761 (1993)] and the absence of these proteins on the cell surface [Dubuisson et al., (1994) supra; Spaete et al., Virology 188: 819-830 (1992)] suggest that initial virion morphogenesis may occur by budding into intracellular vesicles. Thus far, efficient particle formation and release has not been observed in transient expression assays, suggesting that essential viral or host factors are absent or blocked. HCV virion formation and release may be inefficient, since a substantial fraction of the virus remains cell-associated, as found for the pestiviruses. A recent study indicates that extracellular HCV particles partially purifed from human plasma contain complex N-linked glycans, although these carbohydrate moieties were not shown to be specifically associated with E1 or E2 [Sato et al., Virology 196: 354-357 (1993)]. Complex glycans associated with glycoproteins on released virions would suggest transit through the trans-Golgi and movement of virions through the host secretory pathway. If this is correct, intracellular sequestration of HCV glycoproteins and virion formation might then play a role in the establishment of chronic infections by minimizing immune surveillance and preventing lysis of virus-infected cells via antibody and complement.
Genetic variability. As for all positive-strand RNA viruses, the RNA-dependent RNA polymerase (RDRP) of HCV (NS5B) is believed to lack a 3xe2x80x2-5xe2x80x2 exonuclease proof reading activity for removal of misincorporated bases. Replication is therefore error-prone, leading to a xe2x80x9cquasi-speciesxe2x80x9d virus population consisting of a large number of variants [Martell et al., J. Virol. 66: 3225-3229 (1992); Martell et al., J. Virol. 68: 3425-3436 (1994)]. This variability is apparent at multiple levels. First, in a chronically infected individual, changes in the virus population occur over time [Ogata et al., (1991) supra; Okamoto et al., Virology 190: 894-899 (1992)]; and these changes may have important consequences for disease. A particularly interesting example is the N-terminal 30 residue segment of the E2 glycoprotein, which exhibits a much higher degree of variability than the rest of the polyprotein [for examples, see Higashi et al., Virology 197, 659-668. 1993; Hijikata et al., (1991) supra; Weiner et al, (1991) supra]. There is accumulating evidence that this hypervariable region, perhaps analogous to the V3 domain of HIV-1 gp120, may be under immune selection by circulating HCV-specific antibodies [Kato et al., (1993) supra; Taniguchi et al., Virology 195: 297-301 (1993); Weiner et al., (1992) supra. In this model, antibodies directed against this portion of E2 may contribute to virus neutralization and thus drive the selection of variants with substitutions that permit escape from neutralization. This plasticity suggests that a specific amino acid sequence in the E2 hypervariable region is not essential for other functions of the protein such as virion attachment, penetration, or assembly.
Genetic variability may also contribute to the spectrum of different responses observed after IFN-xcex1 treatment of chronically infected patients. Diminished serum ALT levels and improved liver histology, which usually correlates with a decrease in the level of circulating HCV RNA, is seen in xcx9c40% of those treated [Greiser-Wilke et al., J. Gen. Virol. 72: 2015-2019 (1991)]. After treatment, approximately 70% of the responders relapse. In some cases, after a transient loss of circulating viral RNA, renewed viremia is observed during or after the course of treatment. While this might suggest the existence or generation of IFN-resistant HCV genotypes or variants, further work is needed to determine the relative contributions of virus genotype and host-specific differences in immune response.
Finally, sequence comparisons of different HCV isolates around the world have revealed enormous genetic diversity [reviewed in Ref. Bukh et al., (1995) supra]. Because of the lack biologically relevant serological assays such as cross-neutralization tests, HCV types (designated by numbers), subtypes (designated by letters), and isolates are currently grouped on the basis of nucleotide or amino acid sequence similarity. Amino acid sequence similarity between the most divergent genotypes can be a little as xcx9c50%, depending upon the protein being compared. This diversity has important biological implications, particularly for diagnosis, vaccine design, and therapy.
A recent paper [Yoo et al., J. Virol. 69: 32-38 (1995)] reports replication of transcribed HCV-1 RNA after transfection of Huh7 cells. In this paper, T7 transcripts from various derivatives of an HCV-1 cDNA clone were tested for their ability to replicate following transfection of the human liepatoma cell line, Huh7. Possible HCV replication was assessed by strand-specific RT/PCR (using 5xe2x80x2 NTR primers) and metabolic labeling of HCV-specific RNAs with 3H-uridine. Apparently full-length transcripts, terminating with either poly (A) or poly (U), were positive by these assays, but those with a deletion of the 5xe2x80x2 terminal 144 bases were not. In some cultures, HCV-specific RNA was detected in the culture media and this putative virus was used to reinfect fresh Huh7 cells.
The present inventors have been unable to reproduce these results. It appears that this report describes transient replication, rather than authentic HCV infection, with replication and virus production. Some of the data appear self-contradictory. For instance, the positive control reported in this paper was productive transfection of Huh7 cells with RNA extracted from 1 ml of high HCV titer chimpanzee plasma. This extracted sample would contain a maximum of 107 potentially infectious full-length HCV RNA molecules. Under optimum transfection conditions (other than microinjection), greater than 105 RNA molecules of virion RNA (at least for poliovirus, Sindbis virus, or YF) are typically required to initiate a single infectious event. This suggests that in the reported HCV-1 experiment fewer than 100 cells would be productively transfected. Furthermore, at 16 days post-transfection, both positive- and negative-strand RNAs were reportedly detected after eight hours of metabolic labeling. The detection of negative-strand RNA by this method (both for transfected virion RNA and transcript RNA) suggests that HCV is capable of both efficient replication and spread, and that the level of HCV RNA synthesis is similar to that which would be expected for a more robust flavivirus, such as YF (at the peak of a high multiplicity infection). Yet Yoo et al. did not report detection of HCV antigens in these cells using a variety of antisera, nor were they able to report detection of full-length positive- or negative-strands by Northern analysis (which is much more sensitive than metabolic labeling with 3H-uridine). Finally, the critical experiment, demonstrating that RNA or virus derived from the HCV-1 clone is infectious in the chimpanzee model, has not been reported.
Despite the great deal of progress made in the last several years a vast number of questions concerning HCV replication, pathogenesis, and immunity remain unanswered. The field is rapidly reaching a bottleneck where we understand some aspects of the functions of the HCV RNA genome and its encoded proteins, but have no way of experimentally testing structure/function questions in the context of authentic virus replication. Such analyses are critical for understanding each step in the virus life cycle to enable the design of protective vaccines, effective therapy, and HCV diagnostics.
Thus, there is a need in the art for authentic HCV genetic material for expression of infectious HCV RNA.
There is a further need in the art for authentic genetic material for expression of native HCV virions and viral particle proteins, which can, in turn, permit characterization of HCV virion structure.
The art also requires an in vitro culture method for infectious HCV, which would permit analysis of HCV receptor binding, cellular infection, replication, virion assembly, and release.
These and other needs in the art are addressed by the present invention.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention advantageously provides an authentic hepatitis C virus (HCV) DNA clone capable of replication, expression of functional HCV proteins, and infection in vivo and in vitro for development of antiviral therapeutics and diagnostics.
In a broad aspect, the present invention is directed to a genetically engineered hepatitis C virus (HCV) nucleic acid clone which comprises from 5xe2x80x2 to 3xe2x80x2 on the positive-sense nucleic acid a functional 5xe2x80x2 non-translated region (NTR) comprising an extreme 5xe2x80x2-terminal conserved sequence, an open reading frame (ORF) encoding at least a portion of an HCV polyprotein whose cleavage products form functional components of HCV virus particles and RNA replication machinery, and a 3xe2x80x2 non-translated region (NTR) comprising an extreme 3xe2x80x2-terminal conserved sequence, or a derivative thereof selected from the group consisting of adapted virus, live-attenuated virus, replication-competent non-infectious virus, and defective virus. It has been found by the present inventors that various manipulations; effected using genetic engineering techniques, are required to produce an authentic HCV nucleic acid, e.g., a cDNA that can be transcribed to produce infectious HCV RNA. or an infectious HCV RNA. By providing engineered authentic HCV nucleic acids, the present inventors have for the first time enabled dissection of HCV replication machinery and protein activity, and preparation of various HCV derivatives. Previously, since there was uncertainty about whether any given HCV clone contained an error or mutation that led to its inability to function, one could not be certain that starting material for further analysis of HCV was useful or simply due to an artifact. Thus, a major advantage of the present invention is that it provides authentic HCV, thus assuring that any modifications result in real changes rather than artifacts due to errors in the clones provided in the prior art.
A further advantage of the present invention is recognition of the characteristics of an infectious HCV genome, particularly in the polyprotein coding region. In a specific embodiment, the HCV nucleic acid has a consensus nucleic acid sequence determined from the sequence of a majority of at least three clones of an HCV isolate or genotype. Preferably, the HCV nucleic acid has at least a functional portion of a sequence as shown in SEQ ID NO: 1, which represents a specific embodiment of the present invention exemplified herein. It should be noted that while SEQ ID NO: 1 is a DNA sequence, the present invention contemplates the corresponding RNA sequence, and DNA and RNA complementary sequences as well. In a further embodiment, a region from an HCV isolate is substituted for a homologous region, e.g., of an HCV nucleic acid having a sequence of SEQ ID NO: 1. In a further preferred embodiment, exemplified herein, the HCV nucleic acid is a DNA that codes on expression for a replication-competent HCV RNA replicon, or is itself a replication-competent HCV RNA replicon. In a specific example. infra, an HCV nucleic acid of the invention has a full length sequence as depicted in or corresponding to SEQ ID NO:1. Various modifications of the 5xe2x80x2 and 3xe2x80x2 are also contemplated by the invention. For example, the 5xe2x80x2-terminal sequence can be homologous or complementary to an RNA sequence selected from the group consisting of GCCAGCC; GGCCAGCC; UGCCAGCC; AGCCAGCC; AAGCCAGCC; GAGCCAGCC; GUGCCAGCC; and GCGCCAGCC, wherein the sequence GCCAGCC is the 5xe2x80x2-terminus of SEQ ID NO:3.
Still another advantage of the present invention is the demonstration of the importance of the complete 3xe2x80x2-NTR for an infectious HCV clone. The 3xe2x80x2-NTR, particularly the approximately 98 base extreme terminal sequence, which is highly conserved among HCV genotypes, is the subject of U.S. patent application Ser. No. 08/520,678, filed Aug. 29, 1995, which is incorporated herein by reference in its entirety; and PCT International Application No. PCT/US96/14033, filed Aug. 28, 1996, which is also incorporated herein by reference in its entirety. Thus, in a preferred aspect, the functional 3xe2x80x2-NTR comprises a 3xe2x80x2-terminal sequence of approximately 98 bases that is highly conserved among HCV genotypes. In a specific embodiment, the 3xe2x80x2-NTR extreme terminus is homologous or complementary to a DNA having the sequence 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACGGCTAGCTGTGAAAGGTCCGTGAGCCG CATGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCTGATCATGT-3xe2x80x2 (SEQ ID NO:4). In a specific embodiment, exemplified in SEQ ID NO: 1, the 3xe2x80x2-NTR comprises a long poly-pyrimidine region (e.g., about 133 bases); however, alternative length poly-pyrimidine regions are also encompassed, including short regions (about 75 bases), or regions that are shorter or longer. Naturally, in a positive strand HCV DNA nucleic acid, the poly-pyrimidine region is a poly(T/TC) region, and in an positive strand HCV RNA nucleic acid, the poly-pyrimidine region is a poly(U/UC) region.
According to various aspects of the invention, and HCV nucleic acid, including the polyprotein coding region, can be mutated or engineered to produce variants or derivatives with, e.g., silent mutations, conservative mutations, etc. Such clones may also be adapted, e.g., by selection for propagation in animals or in vitro. The present invention further permits creation of HCV chimeras, in which portions of the genome for other genotypes or isolates are substituted for the homologous region of an HCV clone, such as SEQ ID NO:1 or the deposited embodiment, infra. In still other embodiments, the invention provides methods for preparing, and clones comprising, polyprotein coding sequence from an HCV genotype selected from the group consisting of the HCV-1, HCV-1a, HCV-1b, HCV-1c, HCV-2a, HCV-2b, HCV-2c, HCV-3a, and any xe2x80x9cquasi-speciesxe2x80x9d variant thereof. In a further preferred aspect, silent nucleotide changes in the polyprotein coding regions (i.e., variations of the third base of a codon that encodes the same amino acid) are incorporated as markers of specific HCV clones.
In a further aspect of the invention, an HCV nucleic acid, including attenuated and defective variants thereof, can comprise a heterologous gene operatively associated with an expression control sequence, wherein the heterologous gene and expression control sequence are oriented on the positive-strand nucleic acid molecule. In a specific embodiment, the heterologous gene is inserted by a strategy selected from the group consisting of in-frame fusion with the HCV polyprotein coding sequence; and creation of an additional cistron. The heterologous gene can be an antibiotic resistance gene or a reporter gene. Alternatively. the heterologous gene can be a therapeutic gene, or a gene encoding a vaccine antigen, i.e., for gene therapy or gene vaccine applications, respectively. In a specific embodiment where the heterologous gene is an antibiotic resistance gene, the antibiotic resistance gene is a neomycin resistance gene operatively associated with an internal ribosome entry site (IRES) inserted in an SfiI site in the 3xe2x80x2-NTR.
Naturally, as noted above, the HCV nucleic acid of the invention is selected from the group consisting of double stranded DNA, positive-sense cDNA, or negative-sense cDNA, or positive-sense RNA or negative-sense RNA. Thus, where particular secquences of nucleic acids of the invention are set forth, both DNA and corresponding RNA are intended, including positive and negative strands thereof.
An HCV DNA may be inserted in a plasmid vector for translation of the corresponding HCV RNA. Thus, the HCV DNA may comprise a promoter 5xe2x80x2 of the 5xe2x80x2-NTR on positive-sense DNA, whereby transcription of template DNA from the promoter produces replication-competent RNA. The promoter can be selected from the group consisting of a eukaryotic promoter, yeast promoter, plant promoter, bacterial promoter, or viral promoter. In specific examples, infra, phage T7 and SP6 promoters are employed. In a specific embodiment, the present invention is directed to a plasmid clone, p90/HCVFL [long poly(U)], harboring a full-length HCV cDNA which can be transcribed to produce infectious HCV RNA transcripts as deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, USA on Feb. 13, 1997, and assigned accession no. 97879, having a sequence as depicted in SEQ ID NO:5. Naturally, the invention also includes a derivative of this plasmid, selected from the group consisting of a derivative wherein a 5xe2x80x2-terminal sequence is homologous or complementary to an RNA sequence selected from the group consisting of GCCAGCC, GGCCAGCC, UGCCAGCC, AGCCAGCC, AAGCCAGCC, GAGCCAGCC, GUGCCAGCC, and GCGCCAGCC, wherein the sequence GCCAGCC is the 5xe2x80x2-terminus of SEQ ID NO:3; and a derivative wherein a 3xe2x80x2-NTR comprises a short poly-pyrimidine region (since the deposited embodiment has a long poly-pyrimidine region, which may be preferred). In a further embodiment, a derivative of the deposited embodiment may be selected from the group consisting of a derivative produced by substitution of homologous regions from other HCV isolates or genotypes; a derivative produced by mutagenesis; a derivative selected from the group consisting of adapted, live-attenuated, replication competent non-infectious, and defective variants; a derivative comprising a heterologous gene operatively associated with an expression control sequence; and a derivative consisting of a functional fragment of any of the above-mentioned derivatives. Alternatively, portions of the deposited DNA clone, such as the 5xe2x80x2 NTR, the polyprotein coding regions, the 3xe2x80x2-NTR or more generally any coding or non-translated region of the HCV genome, can be substituted with a corresponding region from a different HCV genotype to generate a new chimeric infectious clone, or by extension, infectious clones of other isolates and genotypes. For example, an HCV-1b or -2a polyprotein coding region (or consensus polyprotein coding regions) can be substituted for the HCV-H (1a strain) polyprotein coding region of the deposited clone.
Naturally, the present invention further provides an HCV DNA or RNA transcribed from the full length HCV cDNA harbored in the plasmid clones set forth above.
Thus, the specific HCV genome itself provides an excellent starting material for deriving modified variants of HCV, since any modifications will result from changes to authentic virus, rather than artifacts resulting from an accumulation of changes and errors. The HCV DNA clones or RNAs of the invention can be used in numerous methods, or to derive authentic HCV components, as set forth below.
For example, the invention provides a method for identifying a cell line that is permissive for infection with HCV, comprising contacting a cell line in tissue culture with an infectious amount of HCV RNA, e.g., as produced from the plasmid clones recited above, and detecting replication of HCV in cells of the cell line. Naturally, the invention extends as well to a method for identifying an animal that is permissive for infection with HCV, comprising introducing an infectious amount of the HCV RNA, e.g., as produced by the plasmids above, to the animal, and detecting replication of HCV in the animal. By providing authentic infectious HCV, preferably comprising a dominant selectable marker, the invention further provides a method for selecting for HCV with adaptive mutations that permit higher levels of HCV replication in a permissive cell line or animal comprising contacting a cell line in culture, or introducing into an animal, an infectious amount of the HCV RNA, and detecting progressively increasing levels of HCV RNA in the cell line or the animal. In a specific embodiment, the adaptive mutation permits modification of HCV tropism. An immediate implication of this aspect of the invention is creation of new valid animal models for HCV infection.
The permissive cell lines or animals that are identified using the nucleic acids of the invention are very useful, inter alia, for studying the natural history of HCV infection, isolating functional components of HCV, and for sensitive, fast diagnostic applications, in addition to producing authentic HCV virus or components thereof. As noted above, a particular advantage of the invention is that is represents the first successful preparation of an HCV DNA clone capable of initiating a productive infection in animals or cell lines.
Because the HCV DNA, e.g., plasmid vectors, of the invention encode authentic HCV components, expression of such vectors in a host cell line transfected, transformed, or transduced with the HCV DNA can be effected. For example, a baculovirus or plant expression system can be hamessed to express HCV virus particles or components thereof. Thus, a host cell line may be selected from the group consisting of a bacterial cell, a yeast cell, a plant cell, an insect cell, and a mammalian cell.
Because the invention provides, inter alia, infectious HCV RNA, the invention provides a method for infecting an animal with HCV which comprises administering an infectious dose of HCV RNA, such as the HCV RNA transcribed from the plasmids described above, to the animal. Naturally, the invention provides a non-human animal infected with HCV of the invention, which non-human animal can be prepared by the foregoing methods.
A further advantage of the present invention is that, by providing a complete functional HCV genome, authentic HCV viral panicles or components thereof, which may be produced with native HCV proteins or RNA in a way that is not possible in subunit expression systems, can be prepared. In addition, since each component of HCV of the invention is functional (thus yielding the authentic HCV), any specific HCV component is an authentic component, i.e., lacking any errors that may, at least in part, affect the clones of the prior art. Indeed, a further advantage of the invention is the ability to generate HCV virus particles or virus particle proteins that are structurally identical to or closely related to natural HCV virions or proteins. Thus, in a further embodiment, the invention provides a method for propagating HCV in vitro comprising culturing a cell line contacted with an infectious amount of HCV RNA of the invention, e.g., HCV RNA translated from the plasmids described above, under conditions that permit replication of the HCV RNA.
Naturally, the invention extends to an in vitro cell line infected with HCV, wherein the HCV has a genomic RNA sequence as described above. In a specific embodiment, the cell line is a hepatocyte cell line. The invention further provides various methods for producing HCV virus particles. including by isolating HCV virus particles from the HCV-infected non-human animal of invention; culturing a cell line of the invention under conditions that permit HCV replication and virus particle formation; or culturing a host expression cell line transfected with HCV DNA under conditions that permit expression of HCV particle proteins; and isolating HCV particles or particle proteins from the cell culture. The present invention extends to an HCV virus particle comprising a replication-competent HCV genome RNA, or a replication-defective HCV genome RNA, corresponding to an HCV nucleic acid of the invention as well.
By providing for insertion of heterologous genes in the HCV nucleic acids, e.g., DNA or RNA vectors, the present invention provides a method for transducing an animal susceptible to HCV infection with a heterologous gene, e.g., for gene therapy or gene vaccination, by administering an amount of the HCV RNA to the animal effective to infect the animal with the HCV RNA. In a specific embodiment, such an HCV vector is generated in HCV harbored in the plasmids, described above.
Also provided is an in vitro cell-free assay system for HCV comprising HCV genomic template RNA of the invention, e.g., as transcribed from a plasmid of the invention as set forth above, functional HCV replicase components, and an isotonic buffered medium comprising ribonucleotide triphosphate bases. These elements provide the replication machinery and raw materials (NTPs).
The authentic HCV viral particles and viral particle proteins are a preferred starting material as HCV antigens. Thus, in a further embodiment, the invention provides a method for producing antibodies to HCV comprising administering an immunogenic amount of HCV virus particles to an animal, and isolating anti-HCV antibodies from the animal. Such antibodies may be used diagnostically, e.g., to detect the presence of HCV, or they may be used therapeutically, e.g., in passive immunotherapy. A further method for producing antibodies to HCV comprises screening a human antibody library for reactivity with HCV virus particles of the invention and selecting a clone from the library that expresses an antibody reactive with the HCV virus particle. Naturally, in addition to generating antibodies, the authentic HCV viral particles and proteins of the invention represent preferred starting materials for an HCV vaccine. Preferably, a vaccine of the invention includes a pharmaceutically acceptable adjuvant.
The authentic materials provided herein provide a method for screening for agents capable of modulating HCV replication in vitro and in vivo. Such methods include administering a candidate agent to an HCV infected animal of the invention, and testing for an increase or decrease in a level of HCV infection or activity compared to a level of HCV infection or activity in the animal prior to administration of the candidate agent, wherein a decrease in the level of HCV infection or activity compared to the level of HCV infection or activity in the animal prior to administration of the candidate agent is indicative of the ability of the a agent to inhibit HCV infection or activity. Testing for the level of HCV infection can be performed by measuring viral titer in a tissue sample from the animal; measuring viral proteins in a tissue sample from the animal; or measuring liver enzymes. Alternatively, the HCV genome used to infect the animal may include a heterologous gene operatively associated with an expression control sequence, wherein the heterologous gene and expression control sequence are oriented on the positive-strand nucleic acid molecule, and testing for the level of HCV activity comprises measuring the level of a marker protein in a tissue sample from the animal.
Alternatively, such analysis can proceed in vitro, e.g., by contacting the cell line of claim 32 with a candidate agent; and testing for an increase or decrease in a level of HCV infection or activity compared to a level of HCV infection or activity in a control cell line or in the cell line prior to administration of the candidate agent; wherein a decrease in the level of HCV infection or activity compared to the level of HCV infection or activity in a control cell line or in the cell line prior to administration of the candidate agent is indicative of the ability of the agent to inhibit HCV infection or activity. Testing for the level of HCV infection in vitro can be performed by measuring viral titer in the cells, culture medium, or both; and measuring viral proteins in the cells, culture medium, or both. Alternatively, when the HCV genome used to infect the cell line includes a heterologous gene operatively associated with an expression control sequence, wherein the heterologous gene and expression control sequence are oriented on the positive-strand nucleic acid molecule, and testing for the level of HCV activity comprises measuring the level of a marker protein in a tissue sample from the animal.
A further method for screening for agents capable of modulating HCV replication involves the cell free system described above. This method comprises contacting the in vitro system of the invention with a candidate agent; and testing for an increase or decrease in a level of HCV replication compared to a level of HCV replication in a control cell system or system prior to administration of the candidate agent; wherein a decrease in the level of HCV replication compared to the level of HCV replication in a control cell line or in the cell line prior to administration of the candidate agent is indicative of the ability of the agent to inhibit HCV infection or activity.
The invention includes a method for preparing an HCV nucleic acid comprising joining from 5xe2x80x2 to 3xe2x80x2 on the positive-sense DNA a functional 5xe2x80x2 non-translated region (NTR) comprising an extreme 5xe2x80x2-terminal conserved sequence, a polyprotein coding region encoding HCV proteins that provide for expression of functional HCV proteins, and a 3xe2x80x2 non-translated region (NTR) comprising an extreme 3xe2x80x2-terminal conserved sequence. The method may further comprise determining a consensus sequence for the 5xe2x80x2-NTR, polyprotein coding sequence, and 3xe2x80x2-NTR from a majority sequence of at least three clones of an HCV isolate or genotype. In a specific embodiment, the 3xe2x80x2-NTR comprises an extreme terminal sequence homologous to a DNA having the sequence 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACGGCTAGCTGTGAAAGGTCCGTGAGCCG CATGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCTGATCATGT-3xe2x80x2 (SEQ ID NO:4). In a further specific embodiment, the HCV nucleic acid has a positive strand sequence as depicted in or corresponding to SEQ ID NO:1 comprising substitution of a homologous region from another HCV isolate or genotype.
The present invention also has significant diagnostic implications. In one embodiments the invention provides an in vitro method for detecting antibodies to HCV in a biological sample from a subject comprising contacting a biological sample from a subject with HCV virus particles of the invention, e.g., prepared as described above, under conditions that permit binding of HCV-specific antibodies in the sample to the HCV virus particles; and detecting binding of antibodies in the sample to the HCV virus particles, wherein detecting binding of antibodies in the sample to the HCV virus particles is indicative of the presence of antibodies to HCV in the sample.
An alternative in vitro method for detecting the presence of HCV in a biological sample from a subject comprises contacting a cell line permissive for productive HCV infection with a biological sample, wherein the cell line has been modified to contain a transgene that express a reporter gene product expressed under control of a trans-acting factor produced by HCV; and detecting expression of the reporter gene product, wherein detection of expression of the reporter gene product is indicative of the presence of HCV in the biological sample from the subject. In a related embodiment, the invention provides an in vitro method for detecting the presence of HCV in a biological sample from a subject comprising contacting a cell line permissive for productive HCV infection with a biological sample, wherein the cell line has been modified to contain a defective virus transgene, which defective virus transgene will express a reporter gene product at high levels under control of a trans-acting factor produced by HCV; and detecting expression of the reporter gene product, wherein detection of expression of the reporter gene product is indicative of the presence of HCV in the biological sample from the subject. Thus, a significant advantage of the present invention is in providing permissive (or susceptible) cell lines for these in vitro diagnostics. The method according to claim 64, wherein the defective viral transgene produces an engineered alphavirus, the trans-acting helper factor is alphavirus nsP4 polymerase, and wherein the alphavirus nsP4 polymerase is expressed as a chimeric fusion protein with HCV NS4A, such that the alphavirus nsP4 polymerase-HCV NS4A chimeric fusion protein is cleaved by HCV NS3 proteinase to release functional alphavirus nsP4 polymerase. In the foregoing methods, the biological sample is selected from the group consisting of blood, serum, plasma, blood cells, lymphocytes, and liver tissue biopsy.
In a related aspect, the invention also provides a test kit for HCV comprising authentic HCV virus components, and a diagnostic test kit for HCV comprising components derived from an authentic HCV virus.
Thus, a primary object of the present invention has been to provide a DNA encoding infectious HCV.
A related object of the invention is to provide infectious HCV genomic RNA from DNA clones.
Still another object of the invention is to provide attenuated HCV DNA or genomic RNA suitable for vaccine development, which can invade a cell but fails to propagate infectious virus.
Another object of the invention is to provide in vitro and in vivo models of HCV infection for testing anti-HCV (or antiviral) drugs, for evaluating drug resistance, and for testing attenuated HCV viral vaccines.
Still another object of the invention is to provide for expression of HCV virions or virus particle proteins that can be used to identify the HCV receptor, receptor binding antagonists, and in neutralization assays. In addition, expressed HCV virions or virus particle proteins can be used to develop more effective HCV vaccines, with antigens that are structurally identical to or closely related to native HCV.
A further object of the present invention is to provide HCV diagnostics based on the ability to detect infectious HCV using engineered reporter cells.
Yet another object is to provide authentic viral antigens, particularly viral particles, to assay for HCV-specific antibodies or generate HCV-specific antibodies.
These and other objects of the present invention will be elaborated by the drawings and the Detailed Description of the Invention.